Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models

ABSTRACT Three‐dimensional (3D) cell culture models of solid tumors are currently having a tremendous impact in the in vitro screening of candidate anti‐tumoral therapies. These 3D models provide more reliable results than those provided by standard 2D in vitro cell cultures. However, 3D manufacturing techniques need to be further optimized in order to increase the robustness of these models and provide data that can be properly correlated with the in vivo situation. Therefore, in the present study the parameters used for producing multicellular tumor spheroids (MCTS) by liquid overlay technique (LOT) were optimized in order to produce heterogeneous cellular agglomerates comprised of cancer cells and stromal cells, during long periods. Spheroids were produced under highly controlled conditions, namely: (i) agarose coatings; (ii) horizontal stirring, and (iii) a known initial cell number. The simultaneous optimization of these parameters promoted the assembly of 3D characteristic cellular organization similar to that found in the in vivo solid tumors. Such improvements in the LOT technique promoted the assembly of highly reproducible, individual 3D spheroids, with a low cost of production and that can be used for future in vitro drug screening assays. Biotechnol. Bioeng. 2014;111: 1672–1685. © 2014 Wiley Periodicals, Inc. Liquid Overlay Technique is one of the lowest cost and easy to handle techniques for production of tumor spheroids that mimic the solid tumors formed in vivo. In this work, the authors suggest useful guidelines to perform this in vitro cell culture technique, in order to obtain 3D cell culture models under controlled conditions. The results obtained demonstrate that the optimized parameters promote the formation of multicellular tumor spheroids with reproducible structure and uniform size and shape.